Synthetic PnTx(19) peptide, pharmaceutical compositions and use

ABSTRACT

The present invention relates to a synthetic peptide of 19 amino acids, called PnTx(19), constituted from the sequence of the native toxin PnTx2-6 of the Phoneutria nigriventer spider. It also relates to pharmaceutical compositions containing such a peptide and to the use thereof in the treatment of erectile dysfunction and/or in potentiating the erectile function.

This application is the U.S. national phase of International ApplicationNo. PCT/BR2013/000319, filed 20 Aug. 2013, which designated the U.S. andclaims priority to Brazilian Application No. 1020120208008, filed 20Aug. 2012, and Brazilian Application No. 1020130205745, filed 13 Aug.2013; the entire contents of each of which are hereby incorporated byreference.

The present invention relates to a synthetic polypeptide of 19 aminoacids, called PnTx(19), constituted from the sequence of native toxinPnTx2-6 of the Phoneutria nigriventer spider. It also relates topharmaceutical compositions containing such a peptide and to the usethereof in the treatment of erectile dysfunction and/or in potentiatingthe erectile function.

The venom of the Phoneutria nigriventer spider, popularly known as“aranha-armadeira” (“armed spider”) is rich in bioactive polypeptideswith different pharmacological effects, acting chiefly in ion channelsand receptors. The predominant symptoms caused by its venom result fromgeneral hyper-excitability. Studies have demonstrated that the venomtoxicity comes largely from the effects of the PhTx2 fraction (forreview see: BROGES, M. H. et al. In: Animal Toxin: State of the Art.Perspectives in Health and Biotechnology. Maria Elena de Lima, AdrianoMonteiro de Castro Pimenta, Marie France Martin-Eauclaire, RussolinaBenedeta Zingali and Nerve Rochat (organizing committee), BeloHorizonte-MG. Editora UFMG. 800p. 2009, GOMEZ, M. V. et al. Cellular andMolecular Neurobiology, 22: 5/6, 2002). The toxin PnTx2-6 (MM 5.291.3Da), published from this fraction, reproduces the symptomatic effects ofthe total fraction after intracerebral injection into mice (LE SUEUR, L.P. et al. Acta Neuropathol. 105, 125-134,2003). Its action may beattributed to the delay in the current of inactivation of the channelsfor sodium, having also effect on the activation, in some subtypes ofthese channels, displacing the activation to more negative potentials(MATATEL, A. et al. Biochemistry. 48 (14), 3078-3088, 2009; CASSOLI etal., personal communication, 2012). The effect of PnTx2 on the erectilefunction of rats was described before in the patent applicationP10800596, entitled “Método para a potencialização da função erétilatravees do use das composigoes farmaceuticas de toxina Tx2-6 da aranhaPhoneutria nigriventer” (A method for potentiating the erectile functionby using the pharmaceutical compositions of PnTx2-6 from the spiderPhoneutria nigriventer), potentiates the erection in normotensive rats,restores the erectile function on hypertensive animals (DOCA-sal), andalso in diabetic mice or old-age rats. (for review see: NUNES, K. P. etal. The Journal of Urology, 9(10):2574-81, 2012). These effects deservesto be mediated by the activation of the enzyme Nitric Oxide Synthase(NOS) and release of nitric acid (NO) (YONAMINE, C. M. et al. Toxicon44, 169-172, 2004; NUNES et al., 2008, 2009). Besides, it is suggestedthat some genes involved in the pathway of nitric acid have theirexpression enhanced in the erectile tissue of mice after treatment withthe toxin PnTx2-6 (VILLANOVA, F. E. et al. Toxicon. 54(6), 793-801,2009).

Nitric oxide (NO), a product from the hydrolysis of L-arginine by nitricoxide synthase (NOS), is an essential mediator of the erection process[LEWIS, R. W. et al. A. Definitions, classification, and epidemiology ofsexual dysfunction. In: Sexual Medicine. Sexual Dysfunction in Men andWomen, Lue, T. F.; Basson, R.; Rosen, R.; Giuliano, F.; Khoury, S.;Montorsi, F., (Editores.) International for Sexual Medicine, Paris, p.39-72, 2004]. After release thereof by thenon-adrenergic-non-cholinergic nerves (NANC), nitrergic nerves andendothelial cells, NO spreads into the trabecular and vascular vicinityof the smooth muscle of the penis to stimulate the activity of thesoluble cyclase guanylate (GCs), increasing the concentration of cyclicguanosine monophosphate (GMPc) (TODA, N. et al. Pharmacol. Ther. 106(2), 233-266, 2005; PRIETO, D. Int. J. Impot. Res (20(1), 17-29, 2008).The formation of GMPc induces the decrease in the levels ofintracellular calcium, leading to relaxation of the smooth muscle cellswith a subsequent penile tumescence (TODA et al., 2005). Erection endsdue to the hydrolysis of the GMPc by the enzyme phosphodisesterase type5 ((IGNARRO, L. J. et al. Biochem Biophys Res Commun., 170, 843-50,1990; RAJFER, J. et al. N. Engl. J. Med. 326(2), 90-94, 1992; LEE, M. R.J. Biol. Chem. 272, 5063-5068, 1997; BIVALACQUA, T. J. et al. TrendsPharmacol. Sci., 21 (12), 484-489, 2000). At present, the mainpharmacotherapy used for erectile dysfunction is PDE-5 inhibitors suchas sildenafil (Viagra), taladafil (Cialis® and vardenafil (Levitra®)(MORELAND, R. B et al. Trends Endocrinol. Metab. 10(3), 97-104, 1999;PRIVIERO, F. B. et al. Acta Pharmacol. Sinica 28(6), 751-755, 2007;LEITE, R. at al. Recent Pat. Cardiovasc. Drug Discov. 2(2), 119-132,2007). However, PDE-5 inhibitors are not efficient in the treatment ofpatient with vascular diseases, where the production of NO is impaired.Thus, since the relaxing effect of the smooth muscle of the cavernousbody induced by PnTx2-6 is independent from the inhibition of the enzymePDE-5, this toxin enabling the production of medicaments effective forpatients with erectile dysfunction who may not be treated by thepresently available medicaments.

Another important effect of PnTx2-6 refers to the stimulation of therelease of cerebrocortical synaptosome glutamate of rat [SILVA,2012—personal communication—SILVA, C. N. Análise da liberação deL-glutamato de sinaptosomas de córtex cerebral de rato pela toxinaPnTx2-6 da peconha da aranha armadeira (Phoneutria nigriventer)(Analysis of the release of L-glutamate of from the cerebral cortex ofrats by the toxin PnTx2-6 of the venom of the armed spider (Phoneutrianigriventer)). Initial evaluation of the activity of the syntheticpeptide (PnTx-19) in releasing glutamate and as a potentiator of theerectile function. Master dissertation to be presented on Aug. 31, 2012,at the Biochemistry and Immunology of the Unviersidade Federal de MinasGerais. Belo Horizonte, Minas Gerais, p. 120, 2012]. Yonamine et al.(2005) found that iodized PnTx2-6 can penetrate the hematoencephalicbarrier and thus could exert some effects directly on the CNS, which wasconfirmed by Nunes et al. 2010 (NUNES, K. P. et al. J. Sex Med. 7:3879-88, 2010).

Glutamate (L-glutamate or L-glu) is the main and most abundantexcitatory neurotransmitter of the CNS on mammalians. It exerts acrucial role in mechanisms underlying the synaptic plasticity, which arepart of the physiological bas of behavioral processes such as cognitionand memory. L-glu also performs relevant functions in the development ofthe nervous system like formation, remodeling, elimination of synapses,migration, proliferation, neuronal differentiation, and cell death((PRYBYLOWSKI, K. et al. J. Biol. Chem. 279, 9673-6, 1994; MCKINNEY, R.A. Journal Physiology. 588 (1), 107-116, 2010; MANENT, J. B., REPRESA,A. The Neuroscientist, 13 (3), 268-279, 2007; SCHLETT, K. Current TopicsMedicinal Chemistry. 6(10), 949-960, 2006; VECINO, E. et al. TheInternational Journal of Developmental Biology. 48 (8-9), 965-974,2004). Besides, glutamate can facilitate the penile erection byactivating some cerebra areas that are involved in the control oferectile function line the paraventricular nucleus (PVN), the tegmentedventral area (VTA), the hippocampus, among others (ARGIOLAS, A., MELIS,M. R. Prog. Neurobiol. 47, 235-255, 1995; SONG, Y., RAJASEKARAN, M.Urology. 64(6), 1250-1254, 2004; MELIS, M. R., ARGIOLAS, A. Neuroscienceand Biobehavioral Reviews 35, 939-955, 2011; UCCU, S. et al.Neuropharmacology. 61(1-2), 181-188, 2011).

It is probable that PnTx2-6 has action on this system in the brain,since the first tests for toxicity with this toxin were made viaintracerebral injection, and it was found that it caused erection (forreview see: NUNES et al., 2009). On the other hand, other in vivo testscarried out with the toxin aiming at the study of the potentiation ofthe erectile function were made with subcutaneous or intravenousinjections, which resulted in positive responses (NUNES, 2008b, NUNES etal., 2008a). Besides, Yonamine et al., found that iodized PnTx2-6 canpenetrate the hematoencephalic barrier and thus exert some effectsdirectly on the CNS. Our group also showed that PnTx-6 marked by thetechnetium and injected peripherally, although it concentrates in thepenis, also appears in a small amount in the nervous tissue, making onebelieve that at least a part thereof crosses the hematoencephalicbarrier (NUNES et al., 2010a). Studies on the action of the toxin on thehematoencephalic barrier are in the initial phase, with a view tounderstand the possible entry of this toxin into the CNS. What is knownis that the toxin PnTx2-6 injected by both peripheral and central routesis capable of inducing erection. One should verify the mechanisms bywhich this action takes place. The concrete result of the peripheralaction of the toxin on the erection refers to the relaxation of slicesof the cavernous body when the toxin is applied directly onto this invitro preparation (NUNES et al. 2010a; 2010b; 2012).

Matavel et al. (2009), using molecular modeling tools, indicated a fewamino acids of the PnTx2-6, in unknown epitope, which allegedlyinteracts with the channel for sodium. In this context, the chemicalsynthesis of a partial peptide sequence of the PnTx2-6 that embracesthese amino acids opens new perspectives for structural andpharmacological characterizations of the interaction of the moleculeswith their receptors. Besides, the peptide synthesis, relatively simpleand with possibility of high yield, might provide material necessary tothe pharmacologic study on different tissues, overcoming the limitationobserved with material obtained by purifying the venom or even byheterologous expression, which is difficult and expensive (MATAVEL, A.et al. Biochemistry 48(14), 3078-3088, 2009). Thus, in the presenttechnology one has developed the modified synthetic peptide PnTx(19),which exhibits a short polypeptide chain of 19 amino acids, whichenables one to obtain it easily for commercial purposes, providing thedevelopment of pharmaceutical compositions for use in the treatment oferectile dysfunction and/or in potentiating the erectile function.Besides, in the present technology it was demonstrated that this peptideinduces release of glutamate in cerebrocortical synaptosomes of rats,stimulates the relaxation of slices of cavernous bodies of mice andrats, exhibits lower toxic effect with respect to the native toxin fromwhich it is derived, and further does not exhibit cardiac toxicity,unlike Viagra present on the market. It is also effective inhypertensive animals and exhibits low immunogenicity.

In the prior art, there are new formulations based on polypeptidesderived from spider venom, as disclosed in application WO2009083808,entitled “COM POSITINS AND METHODS FOR TREATING ERECTILE DYSFUNCTION”,which describes compositions comprising a polypeptide isolated from thevenom of the black-widow spider (Latrodectus mactans) and biologicallyactive fragments thereof. However, one did not find any peptide similarto that of the present invention for the treatment of erectiledysfunction.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1—Reversed phase chromatography in HPLC system of the products ofthe synthesis of the peptide PnTx(19). Preparatory column Sphasilpeptide C8 5μ ST 4.6/100, balanced with water Milli-Q 0.1% TFA (byvolume) eluted with a gradient 0 to 25% solution B (0.1%TFA/acetonitrile v/v) in 3 minutes, 25 to 35% of B in 30 minutes and 35to 100% of solution B in 5 minutes, at a flow rate of 2.0 ml/minute.

FIG. 2—Mass spectrometry of the fraction containing the syntheticpeptide PnTx(19). The molecular mass of the PnTx(19) of 2484.9 Da wasdetermined by MALDI-TOF.

FIG. 3—Synaptosomal viability—verified by the activity of the enzymelactate dehydrogenase, which oxidizes the NADH to NAD+. The latterdetermines a reduction in the reading (339 nm), which enables one toqualify the enzymatic activity. The reaction mixture was incubated for60 min with PnTx(19) (10-5 M; 3×10-5 M; 10-6 M; 3×10-6 M) and theactivity percentage evaluated with respect to the lysis triggered by theTriton X-100, considered=100% of lysis. The activity of this enzyme isproportional to the breakage of the synaptosomes, considering that thisis an enzyme of the intracellular compartment.

FIG. 4—Effect of different concentrations of the PnTx(19) in releasingL-glutamate—ELISA Plate containing 300 μl/well of the synaptosomemixture (10% final volume) and NADP+(final concentration of 1 mM) in KRHwith calcium (2 mM), kept at 37° C. under constant stirring wasmonitored. One carried out the reading of the fluorescence on thespectrofluorimeter for 2460 seconds. The enzyme GDH (35 units for thefinal volume of 300 μl) was added to the wells at 60 seconds; at 660seconds, in different wells, one added different concentrations ofPnTx(19) (“10-5M, 3×10-5 M, 10-6 M and 3×10-6 M”) and the KCI (33 mM).As negative controls, one used wells containing untreated synaptosomeswith the toxin. The results exhibit the average and the standard errorof two independent experiments, carried out in triplicate. * P<0.05represent the levels of significance of assays compared with thecontrol—represented by the untreated synaptosomes.

FIG. 5—Effects of the toxin PnTx2-6 and of the peptide PnTx(19)(1×10-6M) on the relaxation of strips of cavernous goodies of mice. Thestrips were pre-contracted with phenylefrine (10-5 M) and relaxed withacetylcholine (10-4M)-control, PnTx2-6 (5×10-8M) and PnTx(19) (x10-6 M)(n=3).

FIG. 6—Effects of the peptide PnTx-19 and of the toxin PnTx2-6 in thecardiac sodium current. A—Effect of the toxin PnTx2-6 on the peak ofsodium current (black arrow) and in the inactivation current (grayarrow). C—Effect of the peptide PnTx-19 on the peak of the sodiumcurrent and in the inactivation current. D—Course of time of peak in thesodium current, during the perfusion of PnTx-19 and PnTx-26. Each blackcircle indicates the amplitude of the Ina, measured every 1 second in amembrane potential of −20 mV. The gray bars indicate the moment when thecell was exposed to PnTx2-6 or PnTX-19 (peptide). The bar graphssummarize the effects of PnTx2-6 500 nM (n=7) and of the peptide PnTx-19(peptide) 1 μM (n=12) in the decay constant (E) and reduction of thesodium current (F). Bars indicate average±SEM, *p<0.05 with respect tothe controls.

FIG. 7—Response dose curve of the peptide PnTx-19 and of the ToxinPnTx2-6 in the right (A) and (B) ventricular pressure, derived±dP/dt (Cand D) and cardiac rate (E) of rats.

FIG. 8—Analysis of the antibody titer. A) Control: adjuvant (aluminumhydroxide). B) antibody titer after administration of the peptidePnTx-19 (10 μG) in different days.

DETAILED DESCRIPTION OF THE TECHNOLOGY

The present invention comprises the peptide PnTx(19),NH3-GERRQYFWIAWYKLANSKK-COOH (SEQ ID NO. 1), synthetized chemically byusing the Fmoc/t-butyl strategy of synthesis on solid support(MERRIFIELD, R. B. Solid-phase peptide synthesis. Adv. Enzymol. Relat.Areas Mol. Biol. (32), 221-296, 1969). The peptide has 19 amino acidresidues, is linear and was designed from the probable three-dimensionalstructure of the PnTx2-6 proposed after analysis for bioinformatics andmolecular modeling studies. This embraces the hydrophobic “core” and thepositively charted residues that surround this region, which, accordingto the molecular modeling studies, are responsible for the interactionof the toxin with the channel for sodium (MATAVEL et al., 2009). Thesequence (discontinuous epitope) developed from these molecular modelingstudies underwent crucial modifications in the present invention, sothat its activity in the erectile function could be kept: the amino acidcysteine, at position 17, was replace by serine, and the peptide wasamidated on the C-terminal portion and acetylated in the N-terminalportion, with a view of increasing the solubility thereof.

The tests for activity demonstrated that PNTx(19), just as PnTx2-6,interferes with the glutamatergic system in cerebrocortical synaptosomesof rats. Different concentrations of the peptide were used, and it wasfound that, in the dose of 3×10-5M, PnTx(19) induced an increase in therelease of glutamate in cerebrocortical synaptosomes of rats (FIG. 4).

The action of the peptide in the erection was verified by using stripsof cavernous body isolated from mice. 1 μM PnTx(19) provided significantrelaxation of the cavernous-body strip of mice, approximately 80% (FIG.5), an action similar to that evoked by 50 nM of PnTx2-6 and 100 μM ofacetylcholine. Probably, the relaxation of the cavernous body is due tothe connection of the linear peptide to the channels for sodium in thecavernous body, triggering the subsequent events in the erection route.

The development of the synthetic peptide PnTx(19) enables one to obtainthe highest amounts of the active molecule with the possibility ofdecreasing the toxic effects with respect to the native toxin PnTx2-6,which provides the development of pharmaceutical compositions for thetreatment of erectile dysfunction and/or in potentiating the erectilefunction.

Such pharmaceutical compositions are characterized by comprising asynthetic peptide and an excipient or a mixture of pharmaceuticallyacceptable excipients, wherein the peptide may be present either in thefree from or coupled to controlled release systems. The controlledrelease systems may include lyposomes, cyclodextrines, biodegradablepolymers, capsules, micro- and nano-capsules, micro- and nano-particles,bolus preparations, osmotic pumps, diffusion devices, lypospheres,transdermal administration systems and/or liquids that, when subjectedto changes in temperature, form a solid or a gel in situ. In this way,the pharmaceutical compositions may be administered by the oral,topical, intramuscular, intravenous, subcutaneous, inhalation routes, orby implantable devices.

The present invention can be better understood with reference to thefollowing examples, which are no limitative of the technology.

Example 1 Chemical Synthesis in Solid Phase and Purification of thePeptide PnTx(19)

The synthetic peptide called PnTx(19) (NH3-GlyGluArgArgGInTyrPheTrpIleAlaTrpTyrLysLeuAlaAsnSerLysLys-OOOH) was partially designed by usingthe bioinformatics program PEPOP. In this way, one proposed adiscontinuous epitope containing 19 amino acids for the toxin PnTx2-6(FLEURY, Cécile, Bioinformatics tools dedicated to the study of thestructure-function-antigenicity relationship in animal peptide toxins.Doctoral thesis, Department of Biochemistry and Immunology of theUnviersidade Federal de Minas Gerais, Belo Horizonte, MG, 180 p., 2009).

With a view to increasing the solubility of the peptide, in the presenttechnology one has synthetized the PnTx(19) with modifications in theN-terminal portion (acetylation) and C-terminal portion (amidation). Theacetylated and amidated peptide PnTx(19) proved to be water-soluble.

The peptide PnTx(19) was purified by reversed-phase chromatography (HPLCÅKTA Explorer 10). The chromatographic profile presented in FIG. 1demonstrates the presence of synthesis by-products and the fractionreferring to the PnTx(19), which elutes with 29% buffer B. Afterpurification, the analysis by mass spectrometry (MALDI-TOF) showed onlya group of molecular species in the spectrum, the molecular mass ofwhich was of 2484.970 Da (FIG. 2), compatible with the mass expected forthe peptide PnTx(19).

Example 2 Test for Synoptosomal Viability

Adult rats (240-300 g) were decapitated the brains were rapidly removed,immersed into homogenization solution (sucrose 0.32 M, EDTA 1 Mm andDithiotreitol (DTT) 0.25 mM, pH=7.4) and kept in ice, then they wereused in preparing the synaptosomes, as previously described by Dunkleyet al 1988 (DUNKLEY, P. R.; Brain Research, 441, 59-71, 1988).

As control of the viability of the synaptosomes and also for verifying apossible lytic activity of the peptide, one carried out tests foractivity of the enzyme lactate dehydrogenase, according to Kubowistz,Ott, 1943 (KUBOWISTZ, F., OTT, C. Biochem Z. 319, 94-117, 1943).

The LDH activity was evaluated in control conditions, in the presence of1% Triton X-100 (100% of lysis) and in the presence of PnTx(19) in thefollowing concentrations 10-5 M; 3×10-5 M; 10-6 M; 3.×10-6 M. Theresults of FIG. 3 demonstrate that the peptide does not affect thesynaptosomal viability and/or exocytotic machinery, since the valuesobtained were 3.4% of lysis for the concentration 10-5 M; 2% of lysisfor the concentration 3×10-5 M; 6% of lysis for the concentration 10-6M; 9% of lysis for the concentration of 3×10-6 M, values comparable withthe control (absence of the peptide).

Example 3 Effects of the Toxin PnTx2-6 and of the Peptide PnTx(19) inCavernous-Body Strips of Mice

Mice were sacrificed by decapitation, their penis were removedsurgically and placed on a Petri dish containing Krebs Ringerbicarbonate—(NaCl, 118.1; KCl, 4.7; KH2PO4, 1.0; MgSo4, 1.0: NaHCO3,25.0; CaCl2, 2.55; and Glucose, 11.1 mM), bubbled with a mixture of 95%O2 and 5% CO2. The gland, the spongy body and the urethra were removedand the cavernous bodies were desiccated with removal of the tunicaalbuginea and separated by cutting the fibrous septum between them. Thestrips of cavernous bodies measuring about 1×1×7 mm were mountedseparately in a chamber, one end thereof being secured to an electrodeand the other linked to a transducer. The chambers contained Krebs (pH7.4) at 37 C, balanced with 95% O2 and 5% CO2.

The tissue was stretched by a passive force of 2.0 mN and stabilized for60 minutes, the solution being replaced every 15 minutes. The changes inisometric force were recorded by using an isometric force transducer(World Precision Instruments, Inc., Sarasota, Fla., USA), connected toan amplifier (TBM-4 model; World Precision Instruments, Inc., USA),using software WinDaq Data Acquisition (Dada® Instruments, USA). Forevaluating the contractile capability of the preparations, a KCIsolution (120 mM) was added to the slices of cavernous bodies and thenthe preparation was washed with Krebs three times.

The slices of cavernous body were pre-contracted with phenylfrin (100-5M) and the relaxation was evoked by acetylcholine 10-4M (control),PntX2-6 (5×10-6M) and the peptide PnTx(19) (1×10-6M).

FIG. 4 shows that the toxin PnTx2-6 (5×10-8 M) and the peptide PnTx(19)(10-6 M) induce relaxation of 83%, while the control with acetylcholine(10-4 M) induced 81.6% of relaxation on strips of cavernous bodies ofmice pre-contracted by phenylfrin (10-5 M). These results indicate thatboth the toxin PnTx2-6 and the peptide PnTx(19) are active in relaxingslices of cavernous bodies of mice.

Example 4 Effect of the Synthetic Peptide PnTx(19) in ReleasingL-glutamate

The release of L-glutamate was analyzed according to the proposal ofNicholls et al. 1987 (NICHOLLS, D. et al. J. Neurochem. 49, 5057, 1987).In order to evaluate whether the peptide was capable of inducing releaseof L-glutamate in cerebrocortical synoptosomes of rats, just as thenative toxin, one analyzed the effect thereof at the followingconcentrations: 10-5M, 3×10-5M, 10-6M, and 2×10-6 M, FIG. 5.

From these results one observes that, in the experimental conditions,only at the concentration of 3×10-5M the peptide was capable of inducingsignificant release of L-glutamate, when compared with the control(absence of PnTx19).

Example 5 Effects of the Peptide PnTx-19 on the Heart

Effect on the Sodium Current

Patch-clamp experiments were carried out with a view to analyze theeffects of the toxin PnTx2-6 and of the peptide PnTx-19 on channels forsodium Nav1.5, present in cardiomyocytes. Records evidenced that thetoxin PnTx2-6 causes a decrease in the peak of sodium current, delayingthe inactivation current. These effects were not observed for PnTx-19(FIGS. 6 A and C). In FIG. 6 B one observes that PnTx2-6 promoted areduction of the density of the sodium current, and this phenomenon wasnot observed in the presence of the peptide either (FIG. 6 D).

Effect on the Ventricular Pressure, Heart Rate and Derivative±dP/dt

The rats were kept under a controlled cycle of 12 h light/darkness at astable temperature with free access to water and food. They weredecapitated 10 to 15 minutes after intraperitoneal injection of Heparin(200 IU). The thorax was opened, the heart was carefully desiccated andperfused through an aortic tip with Krebs-Ringer solution (KRS)containing (by mmol/L) NaCl (118.4), KCl (4.7), KH2PO4 (1.2), MgSO4.7H2O(1.2), CaCl2.2H2O (1.25), glucose (11.7) and NaHCO3 (26.5). Theperfusion flow was kept constant (10 mL/min) at 37° C. and underconstant oxygenation (5% CO2 and 95% O2). A balloon connected to apressure transducer was inserted into the right ventricle, and onemonitored the ventricular pressure, heart rate and derivative±dP/dt(FIG. 7). The balloon volume was adjusted to a final diastolic pressureof about 10 mm Hg. After a period for reaching the balance (30 to 40minutes), 100 μmol/L carrier were injected (control), toxin atconcentrations of 37.8 nmol/L-3.78 μmol/L or a peptide at concentrationsof 37.8 nmol/L-37.8 μmol/L in perfusion buffer.

As shown in FIG. 7, the toxin induced an increase dependent upon theconcentration, under the final right ventricle systolic pressure(RFVSP/PVSFD) and in the +dP/dt, AND −dp/dt, at most by 66.7±13.55%, and61.6±9.89%, respectively (FIG. 7 A; C and D). However, the toxin did notalter the final diastolic pressure and the heart rate. In contrast, thepeptide did not alter any of these parameters, even at highconcentrations (FIGS. 7 B and E).

The results are the average±SEM. Analyses of the one-way variance(ANOVA), followed by the Newman-Keuks post-test were used to analyze theparameters on isolated hearts. For statistical analyses p<0.05 wasconsidered.

Example 6 Immunogenicity of the Peptide PnTx-19

Swiss, male, 20-23 gram mice were divided into 2 groups(Control—Adjuvant: aluminum hydroxide—FIG. 8 A and PnTx-19-10 μG—FIG.8B), each group containing 4 animals. The mice were immunized bysubcutaneous route. 21, 35 and 49 days from the first inoculation, themice received a reinforcement of the above-mentioned preparations. Toverify the presence of antibodies against the PnTx-19, the serumcollected from the mouse tails was evaluated 15, 30, 40 and 60 daysafter the first inoculation through the indirect immunoenzymatic assay(ELISA), as described for other preparations (DEL PINO, F. A. B.,BRANDELLI, A., GONZALES, J. C., HENRIQUES, J. A. P., DEWES, H., Effectof antibodies against β-N-acetylglucosaminidase on reproductiveefficiency of the bovine tick Boophilus microplus. Vet. Parasitol. 79,247-255, 1998).

The immunogenicity of PnTx19 was evaluated after subcutaneous injectionof 10 μg thereof (FIG. 8B). No hypersensitivity or death reactions ofthe animals were observed during the assays. One found a minor increasein the titer of the antibodies in group 2 (PnTx-19-10 μg) 60 days afterthe first inoculation of the peptide (FIG. 8B). It is important to pointout that the dose of peptide of 10 μg is about 12.5 times as high as theDL50 estimated for toxin PnTx2-6 (0.79 μg/mouse) (CORDEIRO M. N., DINIZC. R., VALENTIM A. C., VON EICKSTEDT, GILROY J., RICHARDSON M. Thepurification and amino acid sequences of four Tx2 neurotoxins from thevenom of the Brazilian ‘armed’ spider Phoneutria nigriventer (Keys).FEBS Lett. 310, 153-156, 1992).

The invention claimed is:
 1. A synthetic peptide of comprising thesequence GERRQYFWIAWYKLANSKK (SEQ ID NO: 1).
 2. The synthetic peptide ofclaim 1, wherein the peptide comprises modifications of acetylation andamidation.
 3. A method of using the synthetic peptide of claim 1,comprising administering the synthetic peptide to a subject affected byerectile dysfunction and/or for potentiating erectile function.
 4. Apharmaceutical composition comprising the synthetic peptide of claim 1,and an excipient or a mixture of pharmaceutically acceptable excipients.5. The pharmaceutical composition of claim 4, wherein the peptide ispresent in free form or coupled to a controlled release system selectedfrom the group consisting of lyposomes, cyclodextrins, biodegradablepolymers, capsules, micro- and nanocapsules, micro- and nanoparticles,bolus preparation, osmotic pumps, diffusion devices, lypospheres,transdermal administration systems, liquids that, when subjected tochanges in temperature, form a solid or a gel in situ, and combinationsthereof.
 6. The pharmaceutical composition of claim 4, which isadministered by an oral, topical, intramuscular, intravenous,subcutaneous, or inhalation route, or by an implantable device.
 7. Amethod of treating erectile dysfunction, comprising administering thesynthetic peptide of claim 1 to an individual affected by erectiledysfunction and/or for potentiating erectile function.